Our studies have focused on post-translational modification of chromosomal proteins in the nuclei of Walker-256 carcinosarcoma cells and in the nuclei of other rat tissues. In addition to the previously described modifications of these proteins (acetylation, methylation and phosphomonoester formation), we are concentrating on a new form of phosphorylation which is an acid-labile phosphoryl linkage. We propose to describe in more detail the process by which histones 1 and 4 are phosphorylated on lysine and histidine residues respectively. Experiments to date have demonstrated that only old histone 4 is phosphorylated on histidines, whereas new histone 4 remains free of this modification. Methods required to investigate phosphorus-nitrogen linkages in proteins are primitive, at best, and thus a good deal of our effort is directed toward improving procedures. We are currently investigating high-pressure liquid chromatographic separation of N-phosphoryl amino acids and chemical methods of phosphorylating histidine residues in nucleosomes and histone 4.